The present invention relates generally to the field of biochemical laboratory instrumentation for different applications of measuring properties of samples on e.g. microtitration plates and corresponding sample supports. More particularly the invention relates to more efficient, instrumental features of equipment used as e.g. fluorometers, photometers and luminometers. The applications may be e.g. clinical or research applications.
The routine work and also the research work in analytical biochemical laboratories and in clinical laboratories is often based on different tags or labels coupled on macromolecules under inspection. The typical labels used are different radioactive isotopes, enzymes, different fluorescent molecules and e.g. fluorescent chelates of rare earth metals.
The detection of enzyme labels can be performed by utilizing its natural biochemical function, i.e. to alter the physical properties of molecules. In enzyme immunoassays colourless substances are catalysed by enzyme into colourful substances or non-fluorescent substances to fluorescent substances.
The colourful substances are measured with absorption, i.e. photometric measurement. In the photometric measurement the intensity of filtered and stabilized beam is first measured without any sample and then the sample inside one plate is measured. The absorbance i.e. the absorption values are then calculated.
The fluorescent measurement is generally used for measuring quantities of fluorescent label substance in a sample. The most photoluminescence labels are based on molecular photoluminescence process. In this process optical radiation is absorbed by the ground state of a molecule. Due to the absorption of energy the quantum molecule rises into higher excited state. After the fast vibrational relaxation the molecule returns back to its ground state and the excess energy is released as an optical quantum. Due to losses in this process the average absorbed energies are higher than the average emitted energies. In the following, “activation” is used as a term including excitation of photoluminescence as well as other types of activation by radiation as is described below.
A further measurement method is chemiluminescence measurement where emission of a substance is measured from a sample without activation by illumination. Thus a photoluminometer can also be used as a chemiluminometer.
Further, there is an analysing method called Amplified Luminescent Proximity Homogeneous Assay or AlphaScreen™. The function of the AlphaScreen™ method is based on the use of small beads that attach to the molecules under study. There are two types of beads that are coated with a material acting either as a donor or acceptor of singlet-state oxygen. The measurement starts, when the liquid sample is illuminated by light with wavelength of 680 nm. After this the material in the donor bead converts ambient oxygen into singlet-state oxygen. The single-state molecules have a short lifetime and they can reach only about a 200 nm distance by diffusion in the liquid. If the chemical reaction in question has taken place, both the donor and acceptor beads are bound to the same molecule and so they are close to each other. In this case the singlet-state oxygen may reach the acceptor bead where a series of reactions is started. As the last phase of the reaction the coating material in the acceptor beads emits photons in the 500-700 nm range. If the chemical reaction has not taken place the singlet-state oxygen cannot reach the acceptor bead and the emission light is not detected. By measuring the intensity of light it is possible to conclude the efficiency of the chemical reaction.
The typical instruments in analytical chemical research laboratories are the different spectroscopic instruments. Many of them are utilizing optical region of electromagnetic spectrum. The two common types of instruments are the spectrophotometers and the spectrofluorometers. These instruments comprise usually one or two wavelength dispersion devices, like monochromators. The dispersion devices make them capable to perform photometric, photoluminescence and chemiluminescense measurements throughout the optical spectrum.
Patent document U.S. Pat. No. 6,538,735 describes a prior art device for detecting emission from samples. The principle of the device is illustrated in FIG. 1. In the device 10 the sample is illuminated by high intensity light produced by a light source 12 such as a laser diode. The light transmitted via a fibre bundle 20 activates the sample, which converts the activation light into emission light upon biomolecular binding occurrence. The emitted light is transmitted via a fibre bundle 24 to a detector 41, such as a photomultiplier tube, which detects and measures the amount of light after activation ceases. The fibre bundles that transmit light at the activation and emission wavelength bands are combined such that the common end of the bundle directly above the well includes both fibre types. The fibres may be combined e.g. coaxially. The system can also include a band-pass filter 36 on the emission side, which eliminates extraneous light, including light corresponding to the activation wavelength band. The system can be used in assays based on Amplified Luminescent Proximity Homogeneous Assay technique. The amount of light produced by the sample is proportional to the concentration of an analyte in the sample.
The activation wavelength is between 670 to 690 nm. The light can be generated by employing a high-intensity laser as the activation source, emitting in the preferred wavelength region. The light emitted from the sample has a wavelength band between about 520 nm and 620 nm. This range is at a shorter wavelength than that of the activation wavelength band. The device may include a shutter that prevents light from entering the detector while the laser diode is active, and a filter may prevent light outside the emitted wavelength band from entering the detector.
The emitted signal of the AlphaScreen™ measurement is weak, and the measurement is sensitive to changes in the environment. Therefore it is difficult to achieve an efficient and accurate apparatus for the AlphaScreen™ measurement. Therefore there are certain problems related to the prior art arrangements, especially if several types of measurements are performed with same equipment.
The described prior art arrangement of FIG. 1 uses a coaxial optical cable for transmission and detection. When the cross-section of the cable is used for separate optical wires for activation and detection the usable cross section area is very limited. Therefore both the activation light pulse and the emission light are much attenuated. The attenuation of the activation and emission radiation naturally degrades the efficiency and accuracy of the measurements. The attenuation also causes that the instrument needs more calibration.
One solution could be using a dichroic mirror for separating the optical paths of activation and detection beams as is often used photoluminescence measurements. A prior art arrangement for providing photoluminescence measurements is described e.g. in patent document U.S. Pat. No. 6,071,748. However, there would be further problems if photoluminescence measurement equipment would be used also for AlphaScreen™ measurement. Firstly, if a part of the confocal optics is same for illumination and detection, it can be optimised for only one of these purposes. When a sensitive measurement like AlphaScreen™ is performed it would be important that the optics would be optimised for both illumination and detection. Secondly, different type illumination sources are used in AlphaScreen™ and photoluminescence measurements, and therefore it would be necessary to have optical switches for switching the optical route between two light sources. However, optical switches and the related optics attenuate radiation and therefore decrease the efficiency of the measurements. Good quality optical switches also tend to increase the manufacturing costs of the instrument.
A further significant problem relates to the efficiency of the prior art solutions. In the AlphaScreen™ measurement it is advantageous to use relatively long emission and detection times for each sample. Therefore it takes a long period of time to provide measurements for a whole sample well plate that has a large number of samples. And if several types of measurements are made for the same samples length of the measurement time increases further. The long measurement time naturally means that the throughput of the measurement equipment is not very high. And it also brings a problem that it may be difficult to keep the environmental conditions, such as temperature, sufficiently stable during the measurement of a whole sample assay.
Patent documents U.S. Pat. No. 6,563,584 and U.S. Pat. No. 4,778,763 describe solutions in which a sample is activated with activation light from an excitation light source and then the sample is transported to an optical axis of a detector that measures radiation emitted by the sample. These arrangements are, however, not suitable for such measurements in which an activation of a sample and a detection of radiation emitted by the sample have to be performed simultaneously.